The present invention relates to a method of administering 1) the nucleic acid components identified in defibrotide or the variants thereof, 2) the nucleic acid components identified in defibrotide or the variants thereof in combination with sequence specific oligonucleotides, 3) the nucleic acid components identified in defibrotide or the variants thereof in combination with amino acids or other protein factors, 4) oligonucleotides containing homologous sequences of HIV and cellular regulatory factors or the variants thereof, 5) the nucleic acid components identified in defibrotide or the variants thereof in combination with 4), or 6) sequence non-specific oligonucleotide to treat various disease conditions including HIV infection and its related diseases. The present invention discloses oligonucleotides and vectors which can be used as therapeutic compounds according to the invention. The present invention also relates to a treatment of drug resistance.
Defibrotide is a polyanion salt of a deoxyribonucleic acid obtained from mammalian tissue. Defibrotide is a single-stranded polydeoxyribonucleotide with molecular weight of approximately 20 kDa (low molecular weight form) which may be obtained from bovine lung DNA by controlled hydrolysis. Patents related to its manufacture include U.S. Pat. No. 3,770,720 directed to a process for extracting DNA from mammalian tissue, and U.S. Pat. No. 3,899,481 directed to a process for the controlled partial degradation of DNA extracted from animal organs.
Experimental studies have been performed to investigate the active component of defibrotide. U.S. Pat. No. 3,770,720 discloses that the components of defibrotide include phosphorus 8.5%, Na 9.0%, N 14.0%, deoxyribose 23.2%, total bases 34.0%, guanine 9.4%, thymine 9.4%, adenine 9.2%, cytosine 6.0%, uracil absent, Iodine, and Zinc.
Bracht et al., (Biochem. and Biophys. Res. Com., vol. 200, No. 2, 1994, pp. 933-937) have disclosed four aptamer sequences derived from the unfractionated defibrotide DNA precursor molecule. Two aptamers
(5xe2x80x2-GGTTGGATTGGTTGG-3xe2x80x2 (SEQ ID NO: 1) and 5xe2x80x2-GGTTGGATCGGTTGG-3xe2x80x2) (SEQ ID NO: 2) were identified by thrombin chromatography. Another aptamer
(5xe2x80x2-GGATGGATCGGTTGG-3xe2x80x2) (SEQ ID NO: 3) was found in the PCR product from the double-stranded DNA precursor. The sequence of such aptamer was used to search the EMBL data base and was found in the bovine genome and Angiotensin II-AT1 receptor. The three aptamers were found to have inhibitory activities of thrombin induced platelet aggregation, thromboxane biosynthesis, increase in cytosolic Ca++, and fibrin clot formation. In addition, there is a non-function aptamer (5xe2x80x2GGTGGTGGTTGTGGT3xe2x80x2) (SEQ ID NO: 4) which did not display any of the activities characteristic of defibrotide.
HIV infection is characterized by a progressive decline in immune system function, suppressing the infected host""s ability to overcome other, secondary infection. No cure has been found for HIV infection. The pathogenetic process in HIV infection is never unidimensional but, rather, extremely complex and multifactorial. The pathogenic progression may be only tangentially related to the direct infection of a given target cell. Death is almost inevitable, usually from an overwhelming secondary infection and/or HIV related neoplasm.
Current treatments for HIV infection attempt to retard the progress of the disease or relieve its symptoms. Treatment in use today include certain dideoxynucleotides such as azidothymidine (AZT or zidovudine, Burroughs Wellcome), dideoxyinosine (ddI, Bristol-Myers Squibb) or dideoxycytidine (ddC, Hoffman-LaRoche). These agents can be toxic. Their applicability is limited because of the appearance in some patients of onerous, and sometimes lethal, side effects. These side effects include myelosuppression, peripheral neuropathy, and pancreatitis. In some patients, AZT has lost its effectiveness after prolonged use. While other drugs have been proposed for treatment of HIV infection, including the recent introduction of several HIV protease inhibitors, none have yet been demonstrated to be completely effective. Therefore, there remains a need in the art to develop additional therapeutic agents to treat HIV infection. In particular, there is a need in the art to further identify the active components of defibrotide and their applications in various disease conditions.
It is an object of the invention to provide a method useful in treating a disease condition in a patient, such as infectious diseases, genetic diseases, degenerative diseases, DNA damage, neoplasia, and skin diseases.
To accomplish this objective, the invention provides a method of treatment comprising administering to a patient an effective amount of a therapeutic compound comprising a nucleic acid component of defibrotide, but not including defibrotide.
Preferably, the method is practiced in a marker dependent manner, which method of treating a disease condition comprises:
(a) determining the initial state of a set of disease markers, the disease markers being observable characteristics of a patient which deviate from the normal condition due to the disease state and wherein each disease marker in the set has a predetermined reference range which is indicative of the normal condition,
(b) administering to the patient a dose of a therapeutic compound comprising a nucleic acid component of defibrotide, but not including defibrotide,
(c) screening a panel of second messengers and signal transducers and selecting a repair marker, the intensity of which increases following administration of the therapeutic compound, where intensity is the extent to which the state of the repair marker differs from its state in the normal condition, the repair marker being the concentration of a compound which participates in a cellular regulatory pathway which operates through protein kinase A, protein kinase C, or G-protein,
(d) administering the therapeutic compound at a dose level incrementally higher than the previous dose,
(e) repeating step (d) each time the intensity of the repair marker increases following an incrementally higher dose,
(f) repeating steps (d) and (e) until the intensity of the repair marker in step (c) no longer increases,
(g) administering the therapeutic compound at the highest dose level attained in step (f) until the intensity of the repair marker returns to the normal condition, and
(h) administering the therapeutic compound at a dose level incrementally higher than the previous dose and repeating steps (c), (d), (e), (f) and (g) with one or more additional repair markers until all disease markers of the set of disease markers no longer deviate from the normal condition.
The patient is monitored weekly for three or more weeks. If relapse occurs, as indicated by a deviation of one or more disease and/or repair markers from the normal level, therapy is reinitiated at the highest dose level of the prior course of therapy until normalization is again reached.
In a particularly preferred embodiment of the invention, the method of treating a disease condition comprises the steps of:
(a) determining the initial state of a set of disease markers, the disease markers being observable characteristics of a patient which deviate from the normal condition due to the disease state and wherein each disease marker in the set has a predetermined reference range which is indicative of the normal condition,
(b) administering to the patient a dose of a therapeutic compound comprising a nucleic acid component of defibrotide, but not including defibrotide, wherein the dose of the therapeutic compound is at a level which raises a universal marker to at least five times its normal level, the universal marker being a constitutively expressed molecule which is transcriptionally activated by the therapeutic compound in all disease status, and
(c) continuing to administer the therapeutic compound at the dose level of step (b) until the universal marker returns to its normal level.
The invention also provides a method of treating a disease condition via administering a nucleic acid component of defibrotide with a sequence specific nucleic acids corresponding specifically to selected parts of the viral genome or transcriptional factors.
The invention contemplates treating HIV infection in which HIV is not expressed and wherein the concentration of at least one immunological molecule, such as CD4, CD25, IL-1, IL-3, IL-4, IL-6, TNF and sIL2R, is followed. The method comprises:
(a) administering to the patient an effective amount of a therapeutic compound comprising a nucleic acid component of defibrotide, but not including defibrotide, wherein the effective amount is the amount which causes a universal marker to rise at least five times its normal level, the universal marker being the concentration of a constitutively expressed molecule which is transcriptionally activated by the therapeutic compound in all disease states, and
(b) continuing to administer the effective amount of the therapeutic compound until the universal marker returns to its normal level.
The present invention identifies the active components of defibrotide and the variants thereof. The present invention also provides therapeutic oligonucleotides. Such therapeutic compounds can be used to treat various disease conditions.